Mutations in SPTLC2 gene associated with sensory neuropathy

ABSTRACT

Described are methods and kits for identifying a subject at risk of, or having, a sensory neuropathy related disease, such as sensory neuropathies. In particular, the disclosure is based on the determination of mutations in the SPTLC2 gene causing sensory neuropathies.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/403,619,filed Sep. 17, 2010,the disclosure of which is hereby incorporated herein in its entirety by this reference.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

TECHNICAL FIELD

The present invention relates to a method and kit for identifying a subject at risk of, or having, a sensory neuropathy related disease. In particular, the present invention is based on the determination of mutations in the SPTLC2 gene causing said sensory neuropathies.

BACKGROUND OF THE INVENTION

Hereditary sensory neuropathies (HSNs) form part of the inherited peripheral neuropathies which are generally subdivided into three categories, reflecting the selective or predominant involvement of the motor or sensory peripheral nervous system. The most common variants are the hereditary motor and sensory neuropathies (HMSNs), also called Charcot-Marie-Tooth syndrome, in which both the motor and sensory nerves are affected (Dyck et al. 1993). When only the peripheral motor nervous system is affected, the neuropathy is classified as distal hereditary motor neuropathy (Harding 1993). In contrast, sensory dysfunction prevails in the HSNs. As the autonomic nervous system is involved to a varying degree in HSNs they are often referred to as hereditary sensory and autonomic neuropathies (HSANs) (Dyck 1993).

The HSNs/HSANs are a clinically and genetically heterogeneous group of disorders. Patients usually exhibit prominent distal sensory loss with manifest insensitivity to pain in some. The prominent distal sensory loss frequently leads to chronic ulcerations in feet and hands, sometimes resulting in severe complications such as extensive soft tissue infections, osteomyelitis necessitating amputations of toes and fingers or, in rare instances, even of more proximal parts of the extremities (Dyck 1993). Autonomic dysfunction, such as anhidrosis, fever, blood pressure fluctuations and gastro-intestinal disturbances are present in some patients. Electrophysiologically, axonal nerve damage of sensory neurons is often found, but additional demyelination may also be present (Auer-Grumbach et al. 2003).

HSAN can be transmitted as an autosomal dominant (AD) or autosomal recessive (AR) trait. Isolated patients have also been described (Dyck 1993; Auer-Grumbach 2004). The AD types of HSAN usually present in the second or third decade of life with marked sensory involvement and minimal autonomic and variable motor involvement, while AR HSAN present either as congenital syndromes with striking sensory and autonomic abnormalities or as almost pure autonomic disorders (Verpoorten et al. 2006a).

A classification of the hereditary sensory neuropathies into types HSAN I-V (Dyck, 1993) was made based on age at onset, inheritance pattern and additional features. Molecular genetic research has shown that at least seven genes are associated with the different types of HSNs/HSANs (located on the worldwide web at www.molgen.ua.ac.be/CMTMutations/). Two genes have been associated with AD HSAN: missense mutations in serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) are found in families and individuals with HSAN type I, an adult-onset sensory neuropathy (Bejaoui et al. 2001; Dawkins et al. 2001). Mutations in the small GPTase late endosomal protein RAB7, cause CMT2B (Verhoeven et al. 2003; Meggouh et al. 2006). Mutations in the WNK1/HSN2 gene (protein kinase with-no-lysine(K)-1/hereditary sensory neuropathy type 2) and FAM134B cause AR HSAN type II, an early-onset ulcero-mutilating sensory neuropathy (Lafreniere et al. 2004; Kurth et al. 2009). HSAN type III, also known as Familial Dysautonomia or Riley-Day syndrome, presents with typical prominent autonomic manifestations early in life and is caused by mutations in the inhibitor of kappa-light polypeptide gene enhancer in B cells, kinase complex associated protein (IKBKAP) (Slaugenhaupt et al. 2001). Mutations in neurotrophic tyrosine kinase, receptor type 1 (NTRK1) are reported in families with congenital insensitivity to pain, anhidrosis and mental retardation (CIPA or HSAN type IV) (Indo et al. 1996). HSAN type V, a phenotype closely related to CIPA but with normal mental development and less pronounced anhidrosis, can be caused by mutations in nerve growth factor beta (NGFB) (Einarsdottir et al. 2004) but also by NTRK1-mutations (Houlden et al. 2001; Einarsdottir et al. 2004). Apart from these six HSAN subtypes other forms with distinct additional features exist, e.g., HSAN with gastroesophageal reflux and cough (Kok et al. 2003) and HSAN with spastic paraplegia (Bouhouche et al. 2006b). Recently, the gene for this last form has been identified as cytosolic chaperonin-containing t-complex peptide-1 (CCT5) (Bouhouche et al. 2006a).

The identification of causative genes for the HSAN forms in recent years has provided preliminary insights in the pathogenesis of these rare neuropathies although the fundamental underlying pathomechanisms still remain to be unveiled (Verhoeven et al. 2006). Additional descriptions of HSAN families and patients with known or novel genetic defects are needed to further refine the existing classification and to get a better insight into the molecular basis of these disorders.

SUMMARY OF THE INVENTION

The present invention has identified for the first time a clear link between nucleic acid variations in the SPTLC2 gene and sensory neuropathies. Accordingly, said new genetic markers provide a reliable diagnosis of or prediction of the risk to develop a sensory neuropathy related disease or disorder. Identification of such a genetic variation may not only provide insight as to why the response to treatment varies amongst individuals, but also may potentially decrease morbidity and mortality through improved risk assessment and the administration of personalized medicine.

Accordingly, the present invention provides a method and kit for identifying a subject at risk of, or having, a sensory neuropathy disease, comprising detecting the presence or absence of at least one nucleic acid variant in the SPTLC2 gene, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk of or has a sensory neuropathy disease. Specific regions of interest in the SPTLC2 gene are the coding region of the SPTLC2 gene. The sensory neuropathy disease preferably is a hereditary sensory and autonomic neuropathy disease selected from the group consisting of HSAN type 1, HSAN type 2, HSAN type 3, HSAN type 4 and HSAN type 5.

The methods and kits of the present invention can be carried out in combination with other methods for identifying a subject at risk of, or having, a sensory neuropathy disease. In a preferred embodiment the methods and kits are carried out in combination with a method for the detection of the presence or absence of a nucleic acid variant, or other markers, in any other gene.

Any detection method for the diagnosis and/or prognosis of a sensory neuropathy related disease or disorder forms part of the present invention. Preferred methods and means for the detection of the presence or absence of the nucleic acid variants of the present invention are hybridization, sequencing, PCR, primer extension, MLPA, OLA, restriction site analysis or high-resolution melting analysis for mutation scanning, or a combination thereof.

A further embodiment of the present invention relates to a method for selecting an appropriate treatment or therapeutic agent for a subject at risk of, or having, a sensory neuropathy disease, comprising determining the status of the sensory neuropathy disease by the methods of the present invention and selecting an appropriate treatment or therapeutic agent.

DESCRIPTION OF THE DRAWINGS

FIG. 1. De novo sphingolipid biosynthesis pathway. Left: the canonical pathway with L-serine; right the alternative, disease-related pathway with L-alanine. Condensation with L-alanine instead of L-serine gives rise to a metabolite lacking the C₁ hydroxyl group, obstructing conversion to complex SLs and degradation. The enzymes of the pathway are denoted in green. Myriocin and Fumonisin B1 are mycotoxins inhibiting the enzymes SPT and CerS respectively. SPT: serine palmitoyltransferase; PLP: pyridoxal-5′-phosphate; KSA: 3-keto-sphinganine; CerS: Ceramide synthase; DES: dihydroceramidedesaturase; SO: sphingosine; SL: sphingholipids.

FIG. 2. Missense mutations in SPTLC2 are associated with HSAN-I. (A) Sequence trace files of the G382V mutation in families CMT-1117 (proband indicated by arrow) and CMT-1044. (B) Isolated patient CMT-747.I:1 with the V359M mutation. (C) Patient CMT-635.II:1 carrying a de novo I504F mutation.

FIG. 3. Conservation of mutations among species and structural view of the bacterial SPT enzyme (A) ClustalW multiple protein alignment of the SPTLC2 orthologues from human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), taurus (Bos Taurus), zebrafish (Danio rerio), fly (Drosophila melanogaster), baker's yeast (Saccharomyces cerevisiae) and Gram-negative bacteria with SPT-activity (Sphingomonas paucimobilis). (B) SPT structure of the Sphingomonas paucimobilisSPT homodimer (PDB ID: 2JGT) with the dimeric subunits represented in red and blue. The highlighted amino acids (V246, G268 and G385) correspond to the amino acids (V359, G382 and I504) mutated in the HSAN-I patients (see alignment in panel A).

FIG. 4. In vitro SPT activity measurements of HSAN-I associated SPTLC2 mutants. (A) Fumonisin B1 block assay. SPT activity in HEK293 cells stably expressing wt or mutant SPTLC2 is analyzed by measuring SA accumulation after treatment with Fumonisin B 1. Stable expression of wt SPTLC2 generates an 8.5-fold increase in SPT activity (p=3.24E-5), while the G382V mutant does not increase the SPT activity (p=0.18). The V359M and I504F mutations increase the activity significantly (p=0.00063 and 0.00064, respectively) but not to the same extent as wt SPTLC2. EGFP transfected cells served as control. (B) Radioactive-based SPT activity assay. SPT activity of HEK293 cells stably expressing wt or mutant SPTLC2 was determined by measuring the incorporation of ¹⁴C-labeled L-serine in vitro. Stable expression of wt SPTLC2 results in a significant increase in SPT activity, whereas the expression of G382V fails to raise SPT activity above basal levels. Expression of the V359M or I504F mutant elevates SPT activity, but not as drastically as wt SPTLC2. The right bars represent SPT activity in the presence of the SPT inhibitor myriocin (negative control; see FIG. 1). CPM: Counts per minute. *** P-value<0.001; SA: sphinganine. Data is represented as a mean with error bars representing standard deviations. Error bars and standard deviation were calculated based on three independent experiments.

FIG. 5. Genetic complementation test in S. cerevisiae by tetrad dissection of a heterozygous LCB2/lcb2:: KanMX strain complemented with different YCplac111_LCB2 constructs. Wild-type LCB2 can complement LCB2 deficiency, as shown by the appearance of four equally sized colonies on YPD medium without phytosphingosine at 37° C. The V346M (corresponding to V359M in SPTLC2) and I491F (corresponding to I504F in SPTLC2) LCB2 mutants also rescue the absence of endogenous LCB2. However, yeast transformed with the G369V (corresponding to G382V in SPTLC2) or K366T (dominant negative) mutants yields only colonies when endogenous LCB2 is present, demonstrating the failure of these mutants to complement LCB2 deficiency.

FIG. 6. SPTLC2 mutations affect the enzymatic affinity of SPT. (A) Levels of 1-deoxy-SA in HEK293 cells stably expressing wt or mutant SPTLC2 are measured after an acid and base hydrolysis assay of the extracted lipids. Expression of wt SPTLC2 does not change cellular 1-deoxy-SA levels (p=0.55), whereas all three HSAN-I associated mutants result in significantly elevated 1-deoxy-SA levels (p=0.0025 for V359M; 0.00093 for G382V; 0.00048 for I504F). (B) 1-deoxy-SA levels in HSAN-I patient lymphoblastoid cell lines. The two HSAN-I patients CMT-1044.I:2 (G382V mutation) and CMT-635.II:1 (I504F mutation) show higher levels of 1-deoxy-SA compared to the unaffected parents of CMT-635.II:1 and to two unrelated control individuals. Unfortunately, no lymphoblast cells were available of patient CMT-747.I:1 carrying the V359M mutation. *** P-value<0.001; SA: sphinganine. Data is represented as a mean with error bars representing standard deviations. Error bars and standard deviation was calculated based on three independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term “comprising” is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g., “a” or “an,” “the,” this includes a plural of that noun unless something else is specifically stated.

Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, VoI L CRC Press (1976); Handbook of Biochemistry: Section A Proteins, VoI π, CRC Press (1976).

As used herein, the terms “polypeptide,” “protein,” “peptide” are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

As used herein, the terms “nucleic acid,” “polynucleotide,” “polynucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide.

As used herein, the term “allele” is one of several alternative forms of a gene or DNA sequence at a specific chromosomal location (locus). At each autosomal locus an individual possesses two alleles, one inherited from the father and one from the mother. The term “genotype” means the genetic constitution of an individual, either overall or at a specific locus.

As used herein, the term “homozygous” refers to having two of the same alleles at a locus. The term “heterozygous” refers to having different alleles at a locus.

As used herein, the terms “disorder” and “disease” are used interchangeably.

DETAILED DESCRIPTION

Systematic screening of the known HSAN genes in a large series of patients yielded pathogenic mutations in only 19% of probands (Rotthier et al. 2009), suggesting the involvement of other disease associated genes. By screening a set of functional candidate genes in a large HSAN cohort, the present inventors identified three heterozygous missense mutations in the SPTLC2 subunit of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in the de novo sphingolipid biosynthesis pathway, in four index patients presenting with a typical HSAN type I phenotype. This is particularly surprising since SPTLC2 was previously excluded as a cause for HSAN type I (Dawkins et al. 2002). Moreover, these mutations result in a partial to complete loss of SPT-activity and cause the formation of 1-deoxysphinganine, a neurotoxic metabolite. So, the present findings extend the genetic heterogeneity in HSAN related diseases and enlarge the group of HSAN neuropathies associated with SPT defects.

Thus, according to a first aspect, the invention relates to a method of identifying a subject at risk of, or having, a sensory neuropathy disease, comprising detecting the presence or absence of at least one nucleic acid variant in the SPTLC2 gene or a part thereof, whereby the presence of at least one nucleic acid variant identifies whether a subject is at risk of or has a sensory neuropathy disease.

As used herein, the term “SPTLC2 gene” refers to the gene encoding the second subunit of the serine palmitoyltransferase (SPT). The SPT enzyme is a multisubunit structure, consisting of dimeric subunits of SPTLC1 with either SPTLC2 or SPTLC3 (Hornemann et al. 2007). It is associated with the endoplasmic reticulum (ER) where it catalyzes the pyridixal-5′phosphate (PLP) dependent condensation of L-serine with palmitoyl-CoA. This is the first and rate-limiting step in the de novo biosynthesis of sphingolipids (see also FIG. 1). Sphingolipids are essential components of all eukaryotic cells where they play important roles in membrane structure and in intracellular signaling.

The reference nucleic acid and protein sequences indicated in the current invention are derived from GeneBank (NCBI) and indicated by their respective accession number, as is well known to the person skilled in the art. Frequent updates of the nomenclature for the description of sequence variations are provided on the website of the Human Genome Variation Society. Accordingly, the nucleotide numbering of the coding DNA and RNA reference sequence is as follows: nucleotide +1 is the A of the ATG-translation initiation codon, there is no nucleotide 0, the nucleotide 5′ of the ATG-translation initiation codon is −1. The nucleotide number is preceded by “g.” when a genomic or by “c.” when a cDNA reference sequence is used. Substitutions are designated by “>”. Similarly, the amino acid number is preceded by “p” when a protein reference sequence is used.

The human SPTLC2 gene (serine palmitoyltransferase long chain subunit 2) is located at chromosome 14 at location 14q24.3 and comprises 12 exons. The reference nucleic acid sequence for the human SPTLC2 is NC_000014.8 (gDNA; Version: NC_000014.8 GI:224589805; Region: complement(77973269.78083109); SEQ ID NO:1) or NM_004863 (cDNA; Version: NM_004863.2 GI:31881646; SEQ ID NO:2). The reference protein sequence encoded by the human SPTLC2 gene is NP_004854 (Version: NP_004854.1 GI:4758668; SEQ ID NO:3).

The term “nucleic acid variant” or “polymorphism” or “variant” as used in the present invention, means that the nucleic acid sequence at a certain position in the SPTLC2 gene differs relative to one or more reference nucleic acid sequences. The most simple nucleic acid polymorphism is a polymorphism affecting a single nucleotide, i.e., a single nucleotide polymorphism or SNP. Nucleic acid polymorphisms further include any number of contiguous and/or non-contiguous differences in the primary nucleotide sequence of the nucleic acid under investigation relative to the primary nucleotide sequence of one or more reference nucleic acids. The term “polymorphic position” or “position” refers to the nucleic acid position at which a nucleic acid polymorphism arises. Nucleic acid sequences comprising at least one such polymorphism are referred to as “polymorphic nucleic acid sequences,” “polymorphic polynucleotides,” “polymorphic sequences” or the like. The polymorphism or nucleic acid variant can be an insertion, deletion, substitution, tandem repeat or similar.

The phrase “detecting the presence or absence,” e.g., of a genetic marker as used herein, refers to determining whether or not the relevant genetic event, linked with the occurrence of a disease, is present. In practice, both the absence and the presence of a certain event can function as markers. Accordingly, reference to detecting the presence of a nucleic acid variant generally encompasses determining whether the marker is present, either based on the absence or the presence of the variant in a sample. Moreover, this also includes the possible finding that the marker is not present in the sample, i.e., determining the absence (or presence) of a nucleic acid variant. In both cases determining the presence of the marker can also be done indirectly, e.g., where the presence of a nucleic acid variant is linked to disease, the occurrence of this marker can also be done by determining the homozygous presence of the corresponding allele not comprising the nucleic acid variant. Similarly, allele specific oligonucleotide primers and probes for detecting the presence of a SNP can be specific for the allele where the SNP is not present.

In a specific embodiment, the present invention relates to a method according to the present invention, wherein the SPTLC2 genotype has at least one variant allele of the SPTLC2 gene (heterozygous). In a further embodiment, the method of the invention relates to a method according to the present invention, wherein the SPTLC2 genotype has two variant or wild-type alleles of the SPTLC2 gene (homozygous).

The method of the present invention is particularly suited for the diagnosis and/or prognosis of a sensory neuropathy related disease or disorder in a subject, preferably a human. A sensory neuropathy related disease includes, without limitation, a hereditary sensory neuropathy (HSN), otherwise referred to as hereditary sensory and autonomic neuropathy (HSAN), which can be further classified into HSAN type 1, HSAN type 2, HSAN type 3, HSAN type 4 and HSAN type 5 (see Background section). With the methods of the present invention, the risk for developing a sensory neuropathy disorder can be determined. The “subject” on which the method of the present invention is carried out can be any subject for which the diagnosis/prognosis/risk of an sensory neuropathy needs to be determined. The subject may be a non-human subject such as (but not limited to) a cow, a pig, a sheep, a goat, a horse, a monkey, a rabbit, a dog, a cat, a mouse, a rat, a hamster, a zebrafish, a pufferfish (Fugu), a fly, a worm or C. elegans. More preferably, the subject is a primate. Even more preferably, the subject is a human.

In a further embodiment, the method of the invention comprises the step of determining whether one or more nucleic acid variants in the SPTLC2 gene are present in 0, 1 or 2 copies, more particularly whether a nucleic acid variant in the SPTLC2 gene is present in one or both alleles.

In another embodiment of the above method the presence or absence of the nucleic acid variant can be detected in the SPTLC2 gene or part thereof. Within the present context, “part thereof” refers to the region of interest, i.e., the region of the SPTLC2 gene comprising a nucleic acid variant. More particular, “a part thereof” refers to the 5′UTR, the promoter region, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, exon 12, and/or intron 12. Preferably, the polymorphism is located in the promoter region and/or in the coding region of the SPTLC2 gene, e.g., in at least one of the exons of the SPTLC2 gene. Typically, the nucleic acid variant is, without limitation, a substitution, deletion, insertion, duplication, translocation and/or inversion of at least one nucleotide.

The invention relates in particular to any polymorphism located within the coding region of the SPTLC2 gene as can be identified in the cDNA sequence (SEQ ID NO:2). More particularly, the nucleic acid variant is detected in at least one position of the coding region of the SPTLC2 gene including, without limitation, position 1145, 1075 or 1510 of the cDNA sequence (These positions are relative to the ATG start codon beginning at position 189 of SEQ ID NO: 2. The corresponding positions in SEQ ID NO: 2 are 1333, 1263, and 1698 respectively). More specific, the nucleic acid variant is c.1145G>T, resulting in the amino acid change G382V. Or, the nucleic acid variant is c,1075G>A, resulting in the amino acid change V359M. Or, the nucleic acid variant is c.1510A>T, resulting in the amino acid change 1504F.

As used herein, the term “wild-type” sequence is analogous to the “reference” sequence, and both terms are used interchangeably herein. The reference sequence for e.g., the wild-type human SPTLC2 gene can be the genomic DNA sequence as identified by NC_000014 (gDNA; Version: NC_000014.8 GI:224589805; SEQ ID NO:1) or the cDNA sequence including the coding sequence as identified by NM_004863 (cDNA; Version: NM_004863.2 GI:31881646; SEQ ID NO:2). For example, the allele may be normal as in the reference sequence(s), or it may be a variant, such as a structural or a non-structural variant. The amino acid sequence of the wild-type human SPTLC2 protein is identified by SEQ ID NO:3.

It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the presence or absence of the nucleic acid variants mentioned herein. Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques and may be isolated according to standard nucleic acid preparation procedures well known to those of skill in the art. Many current methods for the detection of allelic variation are reviewed by Nollau et al. (1997), and in standard textbooks, for example, “Laboratory Protocols for Mutation Detection,” Ed. by U. Landegren, Oxford University Press, 1996 and “PCR,” 2^(nd) Edition” by Newton & Graham, BIOS Scientific Publishers Limited, 1997 (incorporated herein by reference).

The method of the present invention can be carried out in vivo or in vitro. Preferred, however, is in vitro detection of nucleic acid variants in the SPTLC2 gene in a biological sample obtained from the subject. The term “biological sample” means a tissue sample or a body fluid sample. A tissue sample includes, but is not limited to, buccal cells, a brain sample, a skin sample, organ sample, placental tissue or fetal cells. The term “body fluid” refers to all fluids that are present in the body including but not limited to blood, plasma, serum, lymph, synovial fluid, amniotic fluid, urine, saliva or cerebrospinal fluid. The biological sample may also be obtained by subjecting it to a pretreatment if necessary, for example, by homogenizing or extracting. Such a pretreatment may be selected appropriately by those skilled in the art depending on the biological sample to be subjected.

A nucleic acid comprising an intended sequence prepared from a biological sample may be prepared from DNA (e.g., gDNA or cDNA) or RNA (e.g., mRNA). Release, concentration and isolation of the nucleic acids from the sample can be done by any method known in the art. Currently, various commercial kits are available such as the QIAamp Blood Kit from Qiagen (Hilden, Germany) for the isolation of nucleic acids from blood samples, or the “High pure PCR Template Preparation Kit” (Roche Diagnostics, Basel, Switzerland) or the DNA purification kits (PureGene, Gentra, Minneapolis, US). Other, well-known procedures for the isolation of DNA or RNA from a biological sample are also available (Sambrook et al., 1989; Ausubel et al., 2003).

When the quantity of the nucleic acid is low or insufficient for the assessment, the nucleic acid may be amplified. Such amplification procedures can be accomplished by those methods known in the art, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, T7- polymerase amplification, and reverse transcription polymerase reaction (RT-PCR).

After performing the extraction and/or amplification procedure, the presence or absence of certain nucleic acid variants in the target sequence can be detected. Numerous methods for detecting a single nucleotide anomaly in nucleic acid sequences are well-known in the art. The present invention is not limited by any particular method used to detect the target sequences disclosed herein. Examples of such methods are described by Gut (2001) and Syvanen (2001), and include, but are not limited to, hybridization methods such as reverse dot blot, Line Probe Assay (LiPA), geneChip™ microarrays, dynamic allel-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, TaqMan™ (5 'nuclease assay) and molecular beacons; allele-specific PCR methods such as intercalating dye, FRET primers and Alphascreen™; primer extension methods such as ARMS, kinetic PCR, SNPstream™, Genetic Bit Analysis™ (GBA), multiplex minisequencing, SNaPshot, pyrosequencing, MassExtend, MassArray, Goodassay, microarray miniseq, APEX (arrayed primer extension), sequence specific priming (SSP), microarray primer extension, Tag arrays, coded microspheres, template-directed incorporation (TDI), fluorescence polarization; oligonucleotide ligation methods such as colorimetric OLA, sequence-coded OLA, multiplex ligation-dependent probe amplification (MLPA), microarray ligation, ligase chain reaction, padlock probes and rolling circle amplification; endonuclease cleavage methods such as restriction site analysis (RFLP) and Invader™ assay; high-resolution melting (HRM) analysis for mutation scanning. In a preferred embodiment, the detection of the presence or absence of a nucleic acid variant is determined by DNA or RNA hybridization, sequencing, PCR, primer extension, MLPA, oligonucleotide ligation assay (OLA), restriction site analysis, or high-resolution melting (HRM) analysis for mutation scanning, or a combination thereof. Accordingly, the method of the present invention optionally comprises the steps of isolating nucleic acids from the sample and/or an amplification step.

The present invention also provides isolated oligonucleotides, i.e., primers and probes, in order to amplify and/or detect nucleic acid variants and/or the wild-type sequence of the SPTLC2 gene. The wild-type sequence of the SPTLC2 gene is identified by its genomic DNA sequence (SEQ ID NO:1) or cDNA sequence (SEQ ID NO:2). Such primers or probes, specifically hybridizing to the target nucleic acid, are of any convenient length such as to consist of at least 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides and up to 40 nucleotides, up to 30 nucleotides or more conveniently up to 25 nucleotides in length, such as, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In general, such primers or probes will comprise nucleotide sequences entirely complementary to the corresponding wild-type or variant locus in the SPTLC2 gene. However, if required one or more nucleotides may be added or one or more mismatches may be introduced, provided that the discriminatory power of the oligonucleotide primer or probe is not unduly affected.

An oligonucleotide primer (or primer pair) designed to specifically recognize and amplify either a wild-type or variant allele at a locus is referred to as an allele specific primer (or primer pair). The same applies for an allele specific probe, i.e., an oligonucleotide probe that specifically hybridizes to either a wild-type or variant allele.

Specific length and sequence of the probes and primers will depend on the complexity of the required nucleic acid target, as well as on the reaction conditions such as temperature and ionic strength. In general, the hybridization conditions are to be stringent as known in the art. “Stringent” refers to the condition under which a nucleotide sequence can bind to related or non-specific sequences. For example, high temperature and lower salt increases stringency such that non-specific binding or binding with low melting temperature will dissolve.

The primers or probes of the invention may carry one or more labels to facilitate detection. The nature of the label is not critical to the invention and may be fluorescent, chemiluminescent, enzymatic, radioactive, chemical or other, provided it doesn't interfere with correct hybridizing of the oligonucleotide.

In a preferred embodiment, the primer or probe consists of 10 to 30 nucleotides, preferably 15 to 30 or 15 to 25 nucleotides, and is capable of specifically forming a hybrid with a part of the SPTLC2 gene and is at least one or more selected from the group consisting of: 1) an oligonucleotide capable of hybridizing under a stringent condition with the sequence as represented by SEQ ID NO:1 or 2, or the complementary thereof; 2) an oligonucleotide of which the sequence is for 80, 85 or 90% identical to the sequence as represented by SEQ ID NO:1 or 2, or the complementary thereof; and 3) an oligonucleotide capable of hybridizing under a stringent condition with the sequence as represented by SEQ ID NO:1 or 2, wherein one or more nucleotides was subjected to a variation such as a substitution, deletion, insertion or addition, or the complementary thereof.

More particular, the present invention relates to an isolated oligonucleotide consisting of 10 to 30 nucleotides, preferably 15 to 30 or 15 to 25 nucleotides, for detecting the presence of one or more nucleic acid variants in SEQ ID NO:1 or 2, or the complementary strand. More specific, the nucleic acid variants are located at position 1145, 1075 or 1510 relative to the ATG start codon beginning at position 189 of SEQ ID NO:2 (The corresponding numbering in SEQ ID NO: 2 is 1333, 1263, and 1698 respectively).

The polymorphism located in the SPTLC2 gene may also be detected in vitro by determining in the isolated SPTLC2 protein the presence or absence of an amino acid change by sequencing said protein. The amino acid change may also be detected by any conventional method known in the art, for example, by mass-spectroscopy, gel electrophoresis, MALDI-TOF mass spectroscopy, ELISA, protein arrays, determination of the molecular weight, or by isoelectrofocusing.

Any human gene can be studied together with the method of the present invention. Of the different genetic markers identified, further important risk factors are polymorphisms or nucleic acid variations in one or more of the following genes (Rotthier et al. 2009): SPTLC1 (e.g., Bejaoui et al. 2001; Dawkins et al. 2001), RAB7A (e.g., Verhoeven et al. 2003; Meggouh et al. 2006), WNK1/HSN2 (e.g., Lafreniere et al. 2004), IKBKAP (e.g., Slaugenhaupt et al. 2001), FAM134B (e.g., Kurth et al. 2009), NTRK1 (e.g., Indo et al. 1996), NGFβ (e.g., Einarsdottir et al. 2004; Houlden et al. 2001), CCT5 (e.g., Bouhouche et al. 2006a), or SCN9A (e.g., Cox et al. 2006).

In a further embodiment, the method of the present invention may also be used in determining whether and which therapeutic agent might be suitable for a patient being at risk of, or having a sensory neuropathy disease. The therapeutic agent may be used to prevent or treat the disease. As used herein, the term “preventing a disease” means inhibiting or reversing the onset of the disease, inhibiting or reversing the initial signs of the disease, inhibiting the appearance of clinical symptoms of the disease. As used herein, the term “treating a disease” includes substantially inhibiting the disease, substantially slowing or reversing the progression of the disease, substantially ameliorating clinical symptoms of the disease or substantially preventing the appearance of clinical symptoms of the disease.

Another aspect of the invention relates to a diagnostic kit for use in the method as described herein. More specific, the invention encompasses a kit for identifying a subject at risk of, at risk of having, or having, a sensory neuropathy disease. This kit can be based on the detection of nucleic acid variants in the SPTLC2 gene of said subject. Accordingly, the kit of the present invention comprises reagents that selectively detect a nucleic acid variant in the SPTLC2 gene.

A kit based on the detection of nucleic acid variants in the SPTLC2 gene may comprise:

-   (a) a means or reagent for detecting the presence or absence of one     or more nucleic acid variants in the SPTLC2 gene of said subject;     and -   (b) optionally, a means for determining, from the nucleic acid     variants detected with the means of step (a), whether the subject is     at risk of, or has, a sensory neuropathy disease.

More preferred, the kit comprises a means for detecting the presence or absence of one or more nucleic acid variants in the coding region of the SPTLC2 gene. In a preferred embodiment of the present invention, the kit comprises:

-   (a) a means or reagent for detecting the presence or absence of a     nucleic acid variant at one or more of the following positions 1145,     1075 or 1510 of the coding region of the SPTLC2 gene, as can be     identified by the cDNA sequence (SEQ ID NO: 2); and -   (b) optionally, a means for determining, from the nucleic acid     variants detected with the means of step (a), whether the subject is     at risk of, or has, an sensory neuropathy disease.

In a specific embodiment the means or reagents in step (a) of said kit may comprise, without limitation:

-   (i) when appropriate, a means for obtaining a target SPTLC2     polynucleic acid present in a biological sample and/or obtaining the     nucleotide sequence thereof; -   (ii) at least one oligonucleotide suitable for detection of a target     SPTLC2 nucleic acid and/or at least one oligonucleotide pair     suitable for amplification of a target SPTLC2 polynucleic acid; -   (iii) when appropriate, an agent for denaturing nucleic acids; -   (iv) when appropriate, an enzyme capable of modifying a double     stranded or single stranded nucleic acid molecule; -   (v) when appropriate, a hybridization buffer, or components     necessary for producing said buffer; -   (vi) when appropriate, a wash solution, or components necessary for     producing said solution; -   (vii) when appropriate, a means for detecting partially or     completely denatured polynucleic acids and/or a means for detecting     hybrids formed in the preceding hybridization and/or a means for     detecting enzymatic modifications of nucleic acids; -   (viii) when appropriate, a means for attaching an oligonucleotide to     a known location on a solid support.

In a preferred embodiment the means or reagent in step (a) of said kit comprises at least one oligonucleotide probe suitable for detection of a target SPTLC2 nucleic acid. In a specific embodiment, the target SPTLC2 nucleic acid is located in the coding region, or part thereof. Even more specific, the target SPTLC2 nucleic acid is located at cDNA position 1145, 1075 and/or 1510 of the SPTLC2 gene. The designated positions either have the wild-type nucleotides or nucleic acid variants thereof. Optionally, the means or reagent in step (a) also includes at least one pair of primers suitable for amplification of a target SPTLC2 polynucleic acid. More particular, the target polynucleic acid is the coding region of the SPTLC2 gene, or part thereof. Even more specific, the target SPTLC2 polynucleic acid comprises cDNA position 1145, 1075 and/or 1510 of the SPTLC2 gene. The designated positions either have the wild-type nucleotides or nucleic acid variants thereof.

The term “hybridization buffer” means a buffer allowing a hybridization reaction between the oligonucleotides and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions. The term “wash solution” means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.

In a specific embodiment of the kit, the means for detecting the presence or absence of nucleic acid variants in the SPTLC2 gene comprises a multiplex assay.

The means in step (b) of said kit, for determining, from the nucleic acid variants in the SPTLC2 gene detected with the means of step (a), whether the subject is at risk of, or has, a sensory neuropathy disease include a table, a chart, or similar, generally referred to as “a predisposition risk algorithm,” indicating the SPTLC2 nucleic acid variants or haplotypes that confer a risk for or the existence of a sensory neuropathy disease. As used herein, the term “chart” refers to graphical presentation, visual aid, diagram, plan, graph, sheet, map or the like including the relevant information. The determination of the risk can be performed manually or with the use of a computer.

The kit of the present invention may include, in addition to the means or reagent for detecting the presence or absence of a nucleic acid variant, a means for detection other risk factors, e.g., nucleic acid variants in a gene, for a sensory neuropathy disease. In a preferred embodiment, the kit additionally includes a means, preferably probes, for detecting the genotype of or a nucleic acid variant in at least one of the genes selected from the group consisting of:SPTLC1, RAB7A, WNK1/HSN2, IKBKAP, FAM134B, NTRK1, NGFβ, CCT5 or SCN9A.

The following examples are intended to promote a further understanding of the present invention. While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

EXAMPLES Example 1 Mutations in SPTLC2 are Associated with HSAN-I

The coding sequence and intron-exon boundaries of SPTLC2 (chromosome location 14q24.3) were analyzed in 78 patients with HSAN, previously screened and found negative for mutations in the other known HSAN genes (SPTLC1,RAB7A, the complete coding region of WNK1/HSN2, FAM134B, NTRK1, NGFB and CCT5) (Rotthier et al. 2009; Kurth et al. 2009). We identified three heterozygous missense mutations in four index patients, for whom clinical and electrophysiological information is summarized in Table 1 and Table 2. The mutations were absent in 300 European control individuals.

A c.1145G>T sequence variation (p.G382V) was found in two families (CMT-1044 and CMT-1117; FIG. 2a ). The proband of family CMT-1117 presented with progressive distal sensory loss and distal muscle weakness in the lower limbs at the age of 38 years. The clinical presentation was similar in a member of family CMT-1044. In addition, this patient experienced dysesthesia in hands and feet and developed osteomyelitis of a thumb. Based on haplotype analysis, these families were found to be unrelated (data not shown).

A second heterozygous mutation (c.1075G>A; p.V359M) was discovered in an isolated patient (CMT-747.I:1; FIG. 2b ). This patient was diagnosed with HSAN after developing distal sensory dysfunction with a foot ulceration necessitating amputation of a toe. No signs of motor or autonomic involvement were noted.

The third mutation (c.1510A>T; p.I504F) is a heterozygous de novo mutation found inpatient CMT-635.II:1 who presented with an atypical early onset sensorimotor neuropathy, complicated with ulcerations, osteomyelitis and anhidrosis (FIG. 2c ). Paternity testing was done to confirm parenthood.

Nerve conduction studies were performed in all patients revealing predominantly axonal sensori-motor neuropathy; this diagnosis was confirmed by a sural nerve biopsy inpatient CMT-747.I:1 (Table 1 and Table 2).

No disease associated sequence variants were identified in the coding region or the intron-exon boundaries of SPTLC3 (chromosome location 20p12.1; GenBank accession number NM_018327).

Example 2 SPTLC2 Mutations are Associated with a Reduction in SPT Activity

All three mutations in SPTLC2 target highly conserved amino acids (FIG. 3a ) rendering it likely that they are functionally important. We set out to investigate the effect of these mutations on SPT activity in stably transfected Flp-in HEK293 cells. The Flp-in system ensures the stable insertion of a single copy of the transgene at a specific genomic location. In this way, moderate and equal expression of the different transgenes is obtained. The cells were treated for 24 h with Fumonisin B1,a mycotoxin that blocks the de novo sphingolipid biosynthesis pathway downstream of SPT (Wang et al. 1991) (FIG. 1). Since condensation of palmitoyl-CoA and serine by SPT is the rate-limiting step in the biosynthesis pathway, the resulting accumulation of sphinganine (SA) reflects the canonical SPT activity (incorporation of L-serine). Stable expression of wild-type (wt) SPTLC2 resulted in an 8-fold increase in SA accumulation compared to control cells stably expressing GFP. This is in agreement with earlier reports, in which overexpression of wt SPTLC2 indeed leads to higher SPT activity (Hornemann et al. 2006). Stable expression of the G382V mutant on the other hand did not increase SA accumulation above basal levels. The V359M and I504F expressing cells showed an increase in SA accumulation but far less pronounced than wt SPTLC2 expressing cells (FIG. 4a ). Thus, the three mutations result in a partial to complete loss of SPT activity.

The effect on canonical SPT activity was confirmed in an alternative radioactive-based in vitro assay. Total lipids were extracted from HEK293 cells stably expressing wt or mutant SPTLC2 and incubated with ¹⁴C-labeled L-serine, PLP and palmitoyl-CoA after which the incorporation of the radioactively labeled serine was measured (FIG. 4b ). The results resembled those of the previous assay. Stable expression of wt SPTLC2 caused a significant increase in SPT activity, whereas the expression of G382V failed to raise SPT activity above basal levels. Expression of the V359M or I504F mutant elevated SPT activity, but not to the same extent as wt SPTLC2. The relative increase in SPT activity in V359M and I504F expressing cells was more pronounced than in the Fumonisin B1 block assay (FIG. 4a ). This difference could be explained by the higher serine concentration used in the latter in vitro assay compared to the serine concentrations present in the cell culture medium during the former assay.

Example 3 SPTLC2 Mutants Differentially Affect in vivo SPT Activity in S. cerevisiae

To corroborate the loss of canonical SPT activity in vivo, we expressed the corresponding yeast mutants (FIG. 3A) in a heterozygous LCB2 deletion yeast strain (LCB2 is the S. cerevisiae orthologue of SPTLC2; the GenBank accession number for the LCB2 sequence is NM_001180370) and performed a tetrad analysis in order to obtain two haploid spores with and two without endogenous LCB2. As expected, all four spores grow at the permissive temperatures of 18° C., regardless of whether they expressed wt or mutant LCB2. At the restrictive temperature (37° C.), spores with (residual) SPT activity will be able to grow, while spores with no or non-functional LCB2 will depend on the external addition of phytosphingosine in order to generate phytosphingolipids and grow (Dunn et al. 2000). Wild-type LCB2 was able to complement the LCB2 deficiency, as apparent from the appearance of four equally sized colonies in the absence of phytosphingosine (FIG. 5). In contrast, but analogous to the dominant negative LCB2 K366T mutation (Gable et al. 2002), yeast spores expressing the G369V mutation (corresponding to G382V in SPTLC2) yielded only colonies when endogenous LCB2 was present, demonstrating the failure of this mutant to complement LCB2 deficiency. The residual activity conferred by the V346M and I491F mutants (corresponding to V359M and I504F respectively in SPTLC2) was sufficient to restore growth at 37° C.; this is in accordance with our biochemical data.

Example 4 Mutant SPT Shows Ambiguity Towards its Amino Acid Substrate

A recent report shows that SPTLC1 mutations in HSAN-I influence the substrate specificity of the SPT enzyme: mutant SPT is able to metabolize L-alanine and to a lesser extent glycine as alternative substrates. This results in the formation of the atypical and neurotoxic sphingoid base metabolites 1-deoxy-SA and 1-deoxymethyl-SA (Zitomer et al. 2009; Penno et al. 2010). The accumulation of these metabolites in the peripheral nerves was postulated to be the underlying cause of HSAN-I (Penno et al. 2010). To study whether SPTLC2 mutations likewise affect the enzymatic affinity of SPT and cause a similar accumulation of these alternative metabolites, the sphingoid base profile of HEK293 cells expressing the mutants was analyzed. In cells stably expressing wt SPTLC2, the amount of 1-deoxy-SA was similar to control cells (FIG. 6a ), showing that an increase in SPT activity as such does not alter substrate specificity. Expression of the mutants on the other hand resulted in up to 20-fold higher 1-deoxy-SA levels compared to control cells, with highest levels in HEK cells stably expressing the G382V or I504F mutant enzyme. The generation of 1-deoxymethyl-SA levels in both HEK cells and lymphoblast cells was below detection limits.

To validate if the results obtained in the HEK cells reflect the situation in HSAN-I patients, 1-deoxy-SA levels in lymphoblast cell lines from two HSAN-I patients, carrying respectively the G382V and I504F mutation, were measured. In both cell lines, accumulation of 1-deoxy-SA was observed when compared to unaffected family members or unrelated healthy control individuals (FIG. 6b ). This finding is in agreement with our in vitro results and more importantly, shows that the accumulation of 1-deoxy-SA could be physiologically relevant.

Materials and Methods to the Examples

Subjects

For this study, a group of 78 patients was selected with hereditary ulcero-mutilating and sensory neuropathies. The inclusion criteria were described previously in Rotthier et al. 2009. The cohort shows a wide variability of clinical features and different modes of inheritance, but all patients share a progressive distal sensory dysfunction. Prior to enrolment in this study, informed consent from all patients or their legal representatives was obtained by the treating physicians.

Mutation analysis

All DNA samples were amplified using the whole genome amplification kit “GenomiPhi V2 DNA Amplification Kit” (GE Healthcare). The coding regions and exon-intron boundaries up to 100 bp up- and downstream of the exons of SPTLC2 and SPTLC3 were PCR-amplified using oligonucleotide primers designed with the Primer3 and SNPbox software tools (Rozen and Skaletsky 2000; Weckx et al. 2004). Primer sequences are listed in Tables 3 and 4. Mutation screening was performed by direct DNA sequencing of purified PCR fragments using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and separated on an ABI3730x1 DNA Analyzer (Applied Biosystems). The resulting sequences were aligned and analyzed with the novoSNP (Weckx et al. 2005) and SeqMan™II programs. Sequence variants were confirmed by repeated PCR on original DNA samples and bidirectional sequencing.

Parenthood was tested using 15 highly informative short tandem repeats (STRs) distributed throughout the genome (ATA38A05, D1S1646, D1S1653, D1S1360, D2S2256, D3S3037, D4S2382, D4S3240, D7S509, D8S1759, D9S1118, D12S1056, D12S2082, D16S2619 and GATA152H04). STRs were PCR-amplified and PCR fragments were loaded on an ABI3730x1 DNA Analyzer. Genotypes were analyzed using Local Genotype Viewer.

Cloning

The SPTLC2cDNA (NM_004863.2) was amplified and cloned into the Gateway® entry vector pDONR221 (Invitrogen) using the primers

SPTLC2_attb1: (SEQ ID NO: 4) 5′-gggacaagtttgtacaaaaaagcaggctatgcggccggagcccggaggctgct-3′; and SPTLC2_attb2: (SEQ ID NO: 5) 5′-ggggaccactttgtacaagaaagctgggtccgtcttctgtttcttcatacgtc-3′.

The SPTLC2 mutations were introduced by site-directed mutagenesis, using the following primers:

SPTLC2_V359M_fw: (SEQ ID NO: 6) 5′-ccacaggccggggtatggtggagtac-3′ SPTLC2_V359M_rv: (SEQ ID NO: 7) 5′-gtactccaccataccccggcctgtgg-3′ SPTLC2_G382V_fw: (SEQ ID NO: 8) 5′-gaacgttcacaaagagttttgttgcttctggaggatatattgg-3′ SPTLC2_G382V_rv: (SEQ ID NO: 9) 5′-ccaatatatcctccagaagcaacaaaactctttgtgaacgttc-3′ SPTLC2_I504F_fw: (SEQ ID NO: 10) 5′-ttcctgccaccccaatttttgagtccagagcc-3′ SPTLC2_I504F_rv: (SEQ ID NO: 11) 5′-ggctctggactcaaaaattggggtggcaggaa-3′.

The constructs were recombined in the destination vectorpEF5/FRT/V5-DEST (Invitrogen), fusing the cDNA with a C-terminal V5-tag. All constructs were validated by sequencing. Stable cell lines were generated using the Flp-in host cell line HEK293 following manufacturer's instructions (Invitrogen).

The yeast LCB2 gene together with its own promotor (700 bp upstream of start codon) and own terminator (450 bp downstream of stopcodon) was cloned into the YCplac111 plasmid vector, harboring a LEU2 gene. Mutations and a HA-tag were introduced with site-directed mutagenesis using the following primers:

LCB2_HA_fw: (SEQ ID NO: 12) 5′-gccactacctgagcccgttgtcagcgtagtctgggacgtcgtatgggtaagcgtagtctggga cgtcgtatgggtaagcgtagtctgggacgtcgtatgggtagacacccctccttattacatttc-3′ LCB2_HA_rv: (SEQ ID NO: 13) 5′-gaaatgtaataaggaggggtgtctacccatacgacgtcccagactacgcttacccatacgacgt cccagactacgcttacccatacgacgtcccagactacgctgacaacgggctcaggtagtggc-3′ LCB2_V346M_fw: (SEQ ID NO: 14) 5′-gcccaactggtcgcggtatgtgtgaaatatttggcg-3′ LCB2_V346M_rv: (SEQ ID NO: 15) 5′-cgccaaatatttcacacataccgcgaccagttgggc-3′ LCB2_G369V_fw: (SEQ ID NO: 16) 5′-gtactttcactaagtcgtttgttgctgctggtggttacattg-3′ LCB2_G369V_rv: (SEQ ID NO: 17) 5′-caatgtaaccaccagcagcaacaaacgacttagtgaaagtac-3′ LCB2_I491F_fw: (SEQ ID NO: 18) 5′-cttatcctgctactccgctgtttgaatcaagagtaagattctg-3′ LCB2_I491F_rv: (SEQ ID NO: 19) 5′-cagaatcttactatgattcaaacagcggagtagcaggataag-3′ LCB2_K366T_fw: (SEQ ID NO: 20) 5′-ctaatgggtactttcactacttcgtaggtgctgctggtg-3′ LCB2_K366T_rv: (SEQ ID NO: 21) 5′-caccagcagcaccaaacgaagtagtgaaagtacccattag-3′

Cell Culture Material and Conditions

HEK293 Flp-in cells were cultivated at 37° C. and 5% CO₂ in DMEM supplemented with 10% fetal bovine serum, L-glutamine and penicillin/streptomycin. Lymphoblastoid cell lines were cultured at 37° C. and 5% CO₂ in RPMI supplemented with 10% fetal bovine serum, L-glutamine, sodium pyruvate and penicillin/streptomycin. All cell culture media and supplements were from Invitrogen.

Lymphoblastoid Cell Lines

Total blood samples were mixed with 15 ml of FicolPaque and centrifuged for 10 min. After washing, lymphocytes were transformed with Epstein-Barr virus and incubated at 37° C. for 2 h. After centrifugation, the pellet was resuspended in 4 ml RPMI complete medium+1% phytohaemagglutinin. Cells were seeded in a 24-well plate and incubated at 37° C. and 5% CO₂ for a minimum of 3 days. Cells were split and supplemented with fresh medium as needed.

Yeast Complementation Assay

The YCplac111 constructs containing wt or mutant LCB2 were transformed (Gietz et al. 2007) in a heterozygous LCB2 deletion strain (BY4743), in which LCB2 has been replaced by a kanamycin resistance gene, and sporulated. The resulting tetrads were dissected to obtain haploid spores which lack endogenous expression of LCB2 and grown on YPD medium with phytosphingosine (15 μM; Avanti Polar Lipids) and 0.1% tergitol at 26° C. After two days, replica plating to different growth media was performed, namely YPD medium at 18° C. and 37° C. (yeast SPT mutants have a thermo-sensitive growth phenotype [Dunn et al. 2000]), synthetic minimal medium without leucine (allowing for selection of transformed spores) and YPD medium with geneticin (selection of LCB2-deficient spores). For each construct, at least six tetrads were analyzed. Unless specified otherwise, media and supplements were from Sigma.

RNA Isolation and mRNA Analysis

Total mRNA was purified using the RNeasy mini kit (Qiagen). DNA inactivation was performed using the Turbo DNA free kit (Ambion) and cDNA synthesis was done with Superscript III first strand synthesis system for RT-PCR (Invitrogen). Expression of SPTLC2 (endogenous and construct) was analyzed using the following primer combinations:

SPTLC2_Fw: (SEQ ID NO: 22) 5′-gagtccagagccaggttttg-3′; and SPTLC2_3′UTR_Rv: (SEQ ID NO: 23) 5′-ctgagggagcaccaaaaag-3′ (for endogenous SPTLC2 expression); or V5_Rv: (SEQ ID NO: 24) 5′-gagagggttagggataggcttac-3′ (for SPTLC2 construct).

Real time qPCR (RT-qPCR) reactions were performed in triplicate with 10 ng cDNA in SYBR Green I mix (Applied Biosystems) and run on ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Primers were validated for specificity and amplification efficiency. RT-qPCR data were normalized according to the method described by Vandesompele et al. 2002. The relative expression levels were used to normalize the data of the Fumonisin B1 block assay, the in vitro SPT activity assay and the 1-deoxy-SA quantification.

Fumonisin B1 Block Assay

This assay was performed as described in Penno et al. 2010. Briefly, Fumonisin B1 (Sigma) was added to the media of exponentially growing cells in a final concentration of 10 μg/ml. As a negative control, the SPT inhibitor myriocin (10 μg/ml, Sigma) was added together with Fumonisin B1. 24 hours after Fumonisin B1 addition, cells were washed twice with PBS, harvested and counted (Coulter® Z2, Beckman Coulter). Next, the cells were subjected to lipid extraction under basic conditions (see below). Sphingoid bases were quantified by LC-MS. Synthetic C 17 sphingosine (Avanti Polar Lipids) was added to each sample as an internal extraction standard.

In Vitro Radioactive-Based SPT Activity Assay

SPT activity was measured using the radioactivity-based assay described by Rütti et al. 2009. In brief, 400 μg total cell lysate, 50 mM HEPES (pH 8.0), 0.5 mM L-serine, 0.05 mM Palmitoyl-CoA, 20 μM Pyridoxal-5′-phosphate, 0.2% sucrose monolaureate (all from Sigma) and 0.1 μCi L-[U-14C] serine (Amersham) were mixed and incubated at 37° C. In the control reaction, SPT activity was specifically blocked by the addition of myriocin (40 μM, Sigma). After 60 min, the reaction was stopped and lipids were extracted according to the method of Riley et al. 1999 (see below).

Lipid Extraction and Hydrolysis

Total lipids were extracted from cells or plasma and extracted according to the method of Riley et al. 1999. For acid hydrolysis, the dried lipids were resuspended in 200 μl methanolic HCl (1N HCl/10M water in methanol) and kept at 65° C. for 12-15 hours. The solution was neutralized by the addition of 40 μl KOH (5M) and subsequently subjected to base hydrolysis, which was performed as follows: 0.5 ml extraction buffer (4 vol. 0.125M KOH in methanol+1 vol. chloroform) was added to the solution. Subsequently, 0.5 ml chloroform, 0.5 ml alkaline water and 100 μl 2M ammonia were added in this order. Liquid phases were separated by centrifugation (12,000 g, 5 min). The upper phase was aspirated and the lower phase washed twice with alkaline water. Finally, the lipids were dried by evaporation of the chloroform phase under N2 and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis.

Extracted lipids were solubilized in 56.7% methanol-33.3% ethanol-10% water and derivatized with ortho-phtalaldehyde. The lipids were separated on a C18 column (Uptispere 120 Å, 5 μm, 125×2 mm, Interchim, France) fluorescence detector (HP1046A, Hewlet Packard) followed by detection on a MS detector (LCMS-2010A, Shimadzu). APCI (atmospheric pressure chemical ionisation) was used for ionization. Non-natural C17 sphingosine (Avanti Polar Lipids) was used as internal standard. Retention times were as follow: C₁₇SO (int.STD): 6 min; sphingosine: 7.5 min; 1-deoxysphingosine: 9 min; 1-deoxymethylsphingosine: 10.5 min; sphinganine: 10.5 min, 1-deoxymethylsphinganine: 13 min; 1-deoxysphinganine: 13.5 min. MS data were analyzed using LCMS solution (Shimadzu) and MS Processor v.11 (ACD Labs).

Statistics

The two-tailed unpaired Student's t-test was used for statistical analysis. Error bars (standard deviation) and p-values (Student t-test) were calculated based on three independent experiments.

TABLE 1 Familial Presenting Disease Patient Origin Mutation history Onset symptoms duration Ulcerations Osteomyellis CMT-747.I:1 Austria c.1075G > A IC 52 y Ulceration and 27 y + (loes) + p.V359M ampulation great toe R CMT- Germany c.1145G > T D 37 y Dysesthesia 35 y − + (thumb R) 1044.I:2 p.G382V and sensory loss distal UL and LL CMT- Austria c.1145G > T D 38 y Sensory loss in  8 y − − 1117.II:1 p.G382V feet CMT- Austria c.1145G > T D ? Asymptomatic ? − − 1117.I:2 p.G382V CMT-535.II:1 Czech c.1510A > T IC (de  5 y Gait difficulties,  9 y + (LL) + Republic p.I504F novo) foot deformities Sensory dys- Autonomic Distal Patient Amputations function dysfunction weakness NCS Additional CMT-747.I:1 + + (distal LL) − − Axonal/ Sural nerve intermediate biopsy: axonal sensorimotor neuropathy in particular of unmyelinated fibers CMT- − + severe − + UL (0- Axonal/ Scoliosis, focal 1044.I:2 distally 3/5) and intermediate epilepsy, Brisk panmodal LL (0/5) sensorimotor Reflexes UL; Claw with hand R > L dysesthesia CMT- − + distally − + LL (2/5) Axonal — 1117.II:1 for touch sensorimotor and vibration CMT- − + distally − + LL (5-/ Axonal Type 2 diabetes 1117.I:2 LL for 5) sensorimotor (onset 71 y) vibration CMT-535.II:1 − + + + (LL) Intermediate — sensorimotor

TABLE 2 Median Ulnar Peroneal Tibial Median Ulnar Sural motor Motor motor motor sensory sensory sensory Patient Age Amp CV Amp CV Amp CV Amp CV Amp CV Amp CV Amp CV Normal values ≧ 4.0 49.0 4.0 49.0 3.0 41.0 3.0 41.0 7.0 46.0 2.0 47.0 1.0 44.0 CMT-747.I:1 79 y R 9.7 44.3 — — 0.1 35.7 — — A A — — — — L 8.4 51.0 — — 0.1 23.3 — — 0.9 35.2 — — — — CMT-1044.I:2 72 y R 0.1 34.0 0.5 37.0 A A A A A A A A A A CMT-1117.II:1 44 y R 6.2 55.0 — — A A A A A A 0.4 38.0 A A CMT-1117.I:2 72 y R 9.9 47.0 5.6 51.0 3.0 42.0 — — — — — — 2.7 33.0 CMT-635.II:1 14 y R 3.8 25.0 2.9 50.0 A A A A A A A A A A L 2.0 29 2.1 53   A A A A — — — — — —

TABLE 3 Exon primers used for PCR and direct sequencing of SPTLC2. SEQ ID SEQ ID Forward primer (5′-3′) NOs Reverse primer (5′-3′) NOs Exon 1 gcagccatttccggtttc 25 ggattgcccagcggatgg 26 Exon 2 ttacaggtgtgagccagtgc 27 tgtgcaaaaatactaagatttc 28 Exon 3 cacaatcttgcacgtaatgaaa 29 cctcagctgctactcctattttg 30 Exon 4 tctgcttccttttgtgtcacc 31 tcagaaaaacaaagcattcttca 32 Exon 5 agtctgaaaaggacacaacaca 33 gctcactctgactgcttttcaa 34 Exon 6 tgatcactgtgctgttgtgc 35 aagactggaccggaagaacat 36 Exon 7 tgaggcatggtttctgaatg 37 tgctgactctgtttccaggt 38 Exon 8 acttcagcctggacaatgga 39 gagcctaaaccagaggcaaa 40 Exon 9 gaccatgttggttgaccttgt 41 gtccatggaaaccacacacc 42 Exon 10 aaatattttatggtgaaatggaaaa 43 tggcatatgtaccaaatgaagg 44 Exon 11 gcctgcatcaccaaagagtt 45 cactgtcaccccctctgtct 46 Exon 12 cctgccgaaggataatcttg 47 gcaaaggaaggattagaagca 48

TABLE 4 Exon primers used for PCR and direct sequencing of SPTLC3. SEQ ID SEQ ID Forward primer (5′-3′) NOs Reverse primer (5′-3′) NOs Exon 1 caaacggtgcagagacc 49 aacccttcataagatgaactcta 50 Exon 2 taacaggagaatgctaacctt 51 cactttagagaggagtaggc 52 Exon 3 agataaccttctacctctgttctaa 53 ttgtcatctagtggccat 54 Exon 4 gaatcgtgcataatcctgg 55 agagacagacacaaggaat 56 Exon 5 aatcttggccttgttgaaa 57 tctaacaaggacctactcaga 58 Exon 6 ctgtccccacaagttgtttt 59 gtcaccttgaagagcagaa 60 Exon 7 tttaggtctgagtgtgaacata 61 tctgtttagctaggaaaggtga 62 Exon 8 ggagggtatttgttagtta 63 ggtgtggtgaactgaattg 64 Exon 9 agggatgggactagatgta 65 gggagattaatgaggcagaa 66 Exon 10 atgcttgccaagttgac 67 cataatctaacgcctgtgc 68 Exon 11 catattccttttttgtcag 69 taaataacccaagagaaac 70 Exon 12 gctattaatctgggctctg 71 ggagaaatccatttatattccttg 72

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What is claimed is:
 1. A method of detecting a mutation in a SPTLC2 gene, the method comprising: (a) obtaining a sample from a subject; (b) contacting a SPTLC2 nucleic acid in a sample from the subject with a detectably labelled oligonucleotide that specifically hybridizes to a SPTLC2 nucleic acid comprising a variant at a position corresponding to a position selected from the group consisting of position 1145, 1075 and 1510 relative to the ATG start codon beginning at position 189 of SEQ ID KO:2, wherein the detectably labelled oligonucleotide comprises i) a nucleotide sequence comprising a variant in a SPTLC2 gene selected from the group consisting of: c.1075G>A, c.1145G>T, and c.1510 A>T, or ii) the complementary strand of the nucleotide sequence of i), and wherein the oligonucleotide hybridizes to the variant SPTLC2 sequence but not the wildtype SPTLC2 sequence; and (c) detecting hybridization of the oligonucleotide with the SPTLC2 nucleic acid under hybridization conditions, and wherein the oligonucleotide will specifically hybridize to the sequence comprising the variant but not to the wildtype SPTLC2 sequence, and wherein detecting hybridization is indicative of a mutation in a SPTLC2 gene.
 2. The method according to claim 1, further comprising detecting the presence or absence of a nucleic acid variant in at least one other gene in the sample from the subject.
 3. The method according to claim 2, wherein the other gene is selected from the group consisting of SPTLC1, RAB7A, WNK1/HSN2, IKBKAP, FAM134B, NTRK1, NGFβ, and CCT5.
 4. The method according to claim 1, wherein the method further comprises use of at least one of the following methods: sequencing, PCR, primer extension, multiplex ligation-dependent probe amplification (MLPA), oligonucleotide ligation assay (OLA), restriction site analysis or high-resolution melting (HRM) analysis for mutation scanning.
 5. The method of claim 1, wherein the variant is c.1075G>A.
 6. The method of claim 1, wherein the variant is c.1145G>T.
 7. The method of claim 1, wherein the variant is c.1510A>T.
 8. The method of claim 1, wherein the detectably labeled oligonucleotide is labelled with a fluorescent, chemiluminescent, enzymatic, radioactive, or chemical label. 